Dahllöf I, Baillie H, Kjelleberg S
School of Microbiology and Immunology and Centre for Marine Biofouling and Bio-Innovation, University of New South Wales, Sydney, New South Wales, Australia.
Appl Environ Microbiol. 2000 Aug;66(8):3376-80. doi: 10.1128/AEM.66.8.3376-3380.2000.
Contemporary microbial community analysis frequently involves PCR-amplified sequences of the 16S rRNA gene (rDNA). However, this technology carries the inherent problem of heterogeneity between copies of the 16S rDNA in many species. As an alternative to 16S rDNA sequences in community analysis, we employed the gene for the RNA polymerase beta subunit (rpoB), which appears to exist in one copy only in bacteria. In the present study, the frequency of 16S rDNA heterogeneity in bacteria isolated from the marine environment was assessed using bacterial isolates from the red alga Delisea pulchra and from the surface of a marine rock. Ten strains commonly used in our laboratory were also assessed for the degree of heterogeneity between the copies of 16S rDNA and were used to illustrate the effect of this heterogeneity on microbial community pattern analysis. The rock isolates and the laboratory strains were also used to confirm nonheterogeneity of rpoB, as well as to investigate the versatility of the primers. In addition, a comparison between 16S rDNA and rpoB PCR-DGGE (denaturing gradient gel electrophoresis)-based community analyses was performed using a DNA mixture of nine isolates from D. pulchra. Eight out of 14 isolates from D. pulchra, all rock isolates, and 6 of 10 laboratory strains displayed multiple bands for 16S rDNA when analyzed by DGGE. There was no indication of heterogeneity for either the rock isolates or the laboratory strains when rpoB was used for PCR-DGGE analysis. Microbial community pattern analysis using 16S rDNA PCR-DGGE showed an overestimation of the number of laboratory strains in the sample, while some strains were not represented. Therefore, the 16S rDNA PCR-DGGE-based community analysis was proven to be severely limited by 16S rDNA heterogeneity. The mixture of isolates from D. pulchra proved to be more accurately described using rpoB, compared to the 16S rDNA-based PCR-DGGE.
当代微生物群落分析经常涉及对16S rRNA基因（rDNA）进行PCR扩增的序列。然而，这项技术存在许多物种中16S rDNA拷贝之间异质性这一固有问题。作为群落分析中16S rDNA序列的替代方法，我们采用了RNA聚合酶β亚基（rpoB）的基因，该基因在细菌中似乎仅以单拷贝形式存在。在本研究中，利用从红藻美丽德尔藻和一块海洋岩石表面分离得到的细菌菌株，评估了从海洋环境中分离出的细菌中16S rDNA异质性的频率。我们还评估了实验室常用的10个菌株中16S rDNA拷贝之间的异质程度，并用于说明这种异质性对微生物群落模式分析的影响。岩石分离菌株和实验室菌株还用于确认rpoB的非异质性，以及研究引物的通用性。此外，使用来自美丽德尔藻的9个分离菌株的DNA混合物，对基于16S rDNA和rpoB的PCR - DGGE（变性梯度凝胶电泳）群落分析进行了比较。通过DGGE分析时，从美丽德尔藻分离出的14个菌株中有8个、所有岩石分离菌株以及10个实验室菌株中的6个在16S rDNA上显示出多条带。当使用rpoB进行PCR - DGGE分析时，无论是岩石分离菌株还是实验室菌株都没有异质性迹象。使用16S rDNA PCR - DGGE进行微生物群落模式分析显示，样本中实验室菌株的数量被高估，而一些菌株未被体现。因此，基于16S rDNA PCR - DGGE的群落分析被证明受到16S rDNA异质性的严重限制。与基于16S rDNA的PCR - DGGE相比，使用rpoB能更准确地描述来自美丽德尔藻的分离菌株混合物。
